Introduction: Oral health is suspected to be linked to heart disease since species of bacteria that cause periodontitis and dental caries have been found in the atherosclerotic plaque in arteries in the heart. Objectives: The aim of this study was to characterize the oral microbiome in patients with coronary artery disease (CAD) and in a patient with dental caries (DC) without any clinical symptoms of CAD. Methods: DNA was extracted from the oral swabs collected from the patients and sequencing was performed by next generation sequencing method using Illumina (MiSeq) platform. The resulting sequencing data set was analysed using QIIME. Results: A total of 31 phyla were found in all the samples. The predominant phylum found in both CAD and DC was Firmicutes (46.09% & 38.98%), Proteobacteria (17.73% & 9.79%), Fusobacteria (13.44% & 17.95%), Bacteroidetes (11.82% & 22.73%), Actinobacteria (8.33% & 7.71%) and TM7 (2.25% & 2.71%). We found a similarity in the bacterial diversity in the two groups of patients. Conclusion: A comparison of the oral microbiome in patients with CAD and DC shows a similarity in the composition of the oral microbiota with variations in the proportion of a few genera.

There is evidence that oral bacteria are linked to a number of systemic diseases including cardiovascular disease (CVD). The oral microbiome has been extensively characterised both by cultivation as well as culture-independent methods. Culture-independent approaches, such as the 16SrRNA gene-based molecular methods, have largely replaced cultivation studies since molecular methods can reveal the identities of currently uncultivated microorganisms. Coronary artery disease (CAD) is characterised by atherosclerotic plaques in the coronary arteries and is the leading cause of mortality in many parts of the world.[1] Although the disease is multifactorial, microorganisms involved in chronic inflammatory processes have been implicated in atherosclerosis development.[2] Metagenomics is the genomic analysis of microorganisms by direct extraction of DNA from an environment/clinical material which avoids the bias imposed by culturing and provides information of microbial diversity including uncultivable microorganisms. 16SrRNA gene sequencing is a good method for studying phylogeny and taxonomy of samples from complex microbiomes.[3] This study aims to characterise the oral microbiome in patients with CAD using 16SrRNA metagenomics.

~ Materials and Methods

Subjects

Four patients with CAD who presented with acute coronary syndrome (ACS) were treated at Department of Cardiology, Rajiv Gandhi Government General Hospital, Chennai. Coronary angiography was done which showed three-vessel CAD in all the cases. One patient with dental caries (DC) without a history of CAD was also included in the study.

Samples

Multiple regions of the oral mucosa were swabbed gently using sterile cotton swabs (HiMedia, India) which were transported to the laboratory in an ice box and frozen at − 80°C until further processing.

DNA extraction and library preparation

DNA was extracted from the oral swabs using Qiagen DNA extraction kit. Four nanograms of DNA (quantified by Nanodrop) was used for amplifying V3–V4 region of 16S region with specific primers. The amplified products were checked by agarose gel electrophoresis. In the next round of PCR (indexing PCR), Illumina sequencing adapters and dual indexing barcodes were added to 25 ng of the first round PCR product using limited-cycle PCR to give a final product of ~580–600 bp. The library was diluted (1:10) and validated for quality by running an aliquot on High Sensitivity Tape Station (Agilent Technologies, USA). All the libraries showed the expected size of ~580–600 bp for V3–V4 region with an effective insert size of ~480 bp flanked on each size by adapters with a combined size of ~140 bp. The libraries were suitable for sequencing on Illumina (MiSeq).

Data analysis

The obtained Illumina paired-end raw reading was quality checked using FastQC. The sequence data of all the samples were analysed using Quantitative Insights Into Microbial Ecology. Rarefaction curves were applied to calculate species richness. Alpha diversity indices were estimated by the Shannon diversity index.

~ Results

The characteristics of the four patients with ACS aged 47–60 years with three-vessel CAD on angiogram and one patient with DC not known to have CAD are shown in [Table 1]. [Figure 1] depicts the rarefaction curve showing greater species richness in the CAD samples, with wide variation between the four CAD samples, and the highest operational taxonomic unit was observed in CAD4. The alpha diversity was higher in CAD samples when compared to the caries sample [Table 2]. The species abundance as well as diversity was greatest in CAD4 [Table 3]. The highest number of unclassified species and genera was also seen in the same sample [Table 4].

A total of 31 phyla such as Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, Actinobacteria, TM7, Spirochaetes, Tenericutes, Cyanobacteria, Chloroflexi, Acidobacteria, SR1, Verrucomicrobia, GN02, Synergistetes, Planctomycetes, Gemmatimonadetes, WWE1, WS3, WPS-2, Thermotogae, (Thermi), OP8, OD1, NKB19, Nitrospirae, Lentisphaerae, Fibrobacteres, Elusimicrobia, Chlorobi and Armatimonadetes were identified in all the samples. Firmicutes was the dominant phylum and found in samples CAD 1 (51.14%), CAD 2 (45.12%), CAD3 (37.26%), CAD4 (50.82%) and DC (38.98%). Bacteroidetes and Fusobacteria were most abundant in the DC sample while Proteobacteria were least abundant [Figure 2]. The same observation was made when we calculated the statistical mean for the four CAD samples and compared with the DC sample. The most predominant phyla found in both groups were Firmicutes (46.09% and 38.98%), Proteobacteria (17.73% and 9.79%), Fusobacteria (13.44% and 17.95%), Bacteroidetes (11.82% and 22.73%), Actinobacteria (8.33% and 7.71%) and TM7 (2.25% and 2.71%) [Figure 3].

In the sample from CAD patients, the predominant genus was Streptococcus (25.27%), Veillonella (10.57%), Leptotrichia (10.46%), Neisseria (6.14%), Prevotella (5.44%), Rothia (4.76%), Capnocytophaga (4.08%), unclassified (3.19%), Fusobacterium (2.84%), Selenomonas (2.63%), Haemophilus (2.49%) and remaining genera accounted for 22.11% of the total microbial population. In the sample from DC patient, the predominant genus was Streptococcus (19.76%), Leptotrichia (14.98%), Capnocytophaga (11.18%), Prevotella (10.63%), Veillonella (10.46%), Selenomonas (4.87%) and remaining genera accounted for 23.88% of the total microbial population.[Figure 4] and [Figure 5]. The total number of genera detected in the CAD samples ranged from 158 to 427, whereas it was 234 in the DC sample [Table 3]. Mannheimia, a genus associated with the absence of caries, was not identified in any of our samples, whereas others such as Neisseria, Cardiobacterium, Rothia, Kingella, and Aggregatibacter were seen in smaller numbers. Streptococcus was the dominant genus in all the samples, whereas Leptotrichia, Capnocytophaga and Prevotella were more abundant in DC when compared to CAD samples. There was no correlation between personal habits such as smoking and diversity or abundance of oral bacterial flora.

The oral cavity may be a potential source of bacteria associated with atherosclerotic plaques. Periodontitis and DC are reported to be associated with increased cardiovascular and peripheral artery disease as determined by clinical disease and angiography. Several mechanisms including specific atherogenic properties of oral bacteria have been postulated.[4] The relationship between alteration of oral microbiome in dental/oral diseases and development of CVD has not been established. Our data confirmed the presence of the five major phyla which constitute the salivary microbiome (Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria), consistent with previous reports.[5] A study by Ismail et al. in 2012 reported no significant qualitative changes of the oral microbiota in patients with atherosclerosis when compared with healthy controls.[6] Although they reported the uncultivated phylum TM7 only in the healthy control group, we found it in both CAD and DC patients. Other studies have shown that the members of the TM7 division are widespread in the oral flora of both healthy and diseased sites and that the subgroup I025 in particular is found primarily at diseased sites.[7] Armingohar et al. have shown significantly higher levels of Prevotella sp. and Capnocytophaga sp. in patients with chronic periodontitis, which was similar to our observation.[3] We detected in addition other species such as Leptotrichia which were more abundant in caries. Majority of the genera which are said to be uniquely associated with the absence of caries such as Neisseria, Cardiobacterium, Rothia, Kingella and Aggregatibacter [8] were seen to be present in both groups of our patients. Mannheimia alone was not identified in any of our samples. Hence, a comparison of patients with CAD and DC shows a similarity in the composition of the oral microbiota with variations in the proportion of a few genera. Patients with periodontal disease share many of the same risk factors as patients with CVD,[9] suggesting that both diseases share common etiological pathways. This relationship may be mediated by alterations in the oral microbiome. The limitation of our study was the small sample size; nevertheless, it shows a similarity in the bacterial diversity in the two groups of patients. Preliminary analysis of the data in these patients suggests a similarity between the oral microbiome in patients with CAD and DC.

Financial support and sponsorship

This study has been carried out as part of the research program under MRU and the publication fee is met under the contingencies of the MRU, Department of Health Research.